DK175819B1 - LAV virus variants, DNA and protein components thereof, and uses thereof, in particular for diagnostic purposes and for the preparation of immunogenic preparations - Google Patents

Description

in DK 175819 B1

The present invention relates to viruses capable of inducing lymphadenopathy (hereinafter referred to as the abbreviation LAS), acquired immunodeficiency syndrome (hereinafter referred to as the abbreviation AIDS), the antigens of the viruses, particularly in purified form, and methods. for the production of these antigens, especially anti-5 genes from these virus envelopes. The invention also relates to polypeptides, either glycosylated or non-glycosylated, encoded by the DNA sequences.

The invention also relates to cloned DNA sequences which can be hybridized to genomic RNA and DNA from the novel lymphadenopathy-associated viruses (LAVs) described hereinafter, methods of producing them and using them. In more detail, the stable probes containing a DNA sequence can be used to detect the novel LAV viruses or related viruses or DIMA proviruses in any medium, especially biological, or samples containing any any of these.

15

For the human retrovirus, an important genetic polymorphism has been observed at the site of acquired immunodeficiency syndrome (AIDS) and other diseases such as lymphadenopathy syndrome (LAS), AlDS-related complex (ARC), and presumably some encephalopathies (see Weiss, 1984). All of the isolates analyzed up to now 20 also have different restriction maps, even when they emerge from the same site and at the same time (Benn et al., 1985). Only identical restriction maps have been observed for the first two isolates, termed lymphadenopathic-associated virus, LAV (Alizon et al., 1984), and human T-cell lymphotrophic virus type 3, HTLV-3 (Hahn et al. , 1984), and therefore appear as an exception.

The genetic polymorphism of the AIDS virus could be better assessed after determining the complete nucleotide sequences of LAV (Wain-Hobson et al., 1985), HTLV-3 (Ratner et al., 1985; Muesing et al., 1985), and a third. isolate, designated AlDS-associated retrovirus, ARV (Sanchez-Pescador et al., 1985). In addition to the nucleic acid variations responsible for restriction map polymorphism, in particular, isolates could differ significantly at the protein level, especially in the envelope (up to 13% difference between ARV and LAV), both in amino acid substitutions and in reciprocal insertions deletions. (Rabson and Martin, 1985).

DK 175819 B1 2

Nevertheless, the above-mentioned differences do not go so far as to destroy a level of immunological activity which is sufficient, as demonstrated by similar proteins, i.e. nuclear proteins of a similar nature such as the p25 proteins, or similar envelope glycoproteins such as the 110-120 kd glycoproteins, ability to cross-react immunologically. Thus, the proteins of any of the LAV viruses can be used for the in vitro detection of antibodies induced in vivo and present in biological fluids of individuals infected with the other LAV variants. Therefore, these viruses are grouped into a class of LAV viruses, which are then generally said to belong to the class of LAV-1 viruses.

10

The present invention is based on the observation of novel viruses which, although considered to be responsible for diseases clinically related to AIDS and still belonging to the class of "LAV-1 viruses", are far greater extent differs genetically from the aforementioned LAV variants.

15

The novel viruses are basically characterized by the DNA sequences shown in FIG. 7A-7J (LAVEU) and FIGS. 8A-8I (LAVMAl) ·

The invention further relates to variants of the novel viruses whose RNA molecules or the related cDNA molecules derived from the RNA molecules are hybridizable to corresponding portions of either LAVeu or LAVmal's cDNA molecules.

The invention also relates to the DNA molecules of the viruses themselves, including 25 DNA fragments derived therefrom and hybridizable to either LAVeli or LAVmal's genomic RNA. In particular, the DNA molecules consist of the cDNA molecules or cDNA fragments mentioned above or of recombinant DNA molecules containing the cDNA molecules or cDNA fragments.

30

The invention further relates to DNA recombinants containing DNA molecules or cDNA fragments from either LAVel, or LAVmal, or from related viruses. It is, of course, clear that fragments which include certain deetions or mutations that do not substantially alter their ability to hybridize with LAVEU 35 or LAVMA | retroviral genomes as well are to be readily apparent. valents to the DNA molecules or DNA fragments discussed more specifically above.

More specifically, the invention also relates to cloned probes that can be prepared from any DNA fragment of the present invention, thus recombinant DNA molecules containing such fragments, in particular any plasmid that can be amplified in prokaryotic or eukaryotic cells, and carrying these fragments.

LAV virion RNA can be detected directly, for example, in the blood, body fluids, and blood products (e.g., in the anti-hemophilic factors such as Factor VIII concentrates) using the cloned DNA containing a LAVfu or LAVMAL DNA fragment as molecular hybridization probe. - either by labeling with radionucleotides or with fluorescent reagents. A convenient method of obtaining this detection comprises that viruses are immobilized on a substrate, e.g., nitrocellulose filters, etc., that the virion is degraded and hybridized with labeled (radiolabelled or "cold" fluorescence or enzyme-labeled) probes. Such a method has already been developed for peripheral blood hepatitis B virus (according to J. Scotto et al., In Hepatology (1983), 3, 379-384).

20

Probes according to the invention can also be used for rapid screening of genomic DNA derived from the tissue of patients with LAV-related symptoms to see if the proviral DNA or RNA present in host and other tissues is related to LAVEU or LOW template.

25

A method that can be used for such screening includes the following steps: extraction of DNA from tissue, restriction enzyme cutting of the DNA, electrophoresis of the fragments and Southern blotting of genomic DNA from tissue, subsequent hybridization with labeled cloned LAV proviral DNA. In vivo hybridization can also be used.

Lymphatic fluids and tissues and other non-lymphoid tissues from humans, primates, and other mammalian species may also be screened to see if other evolutionarily related retroviruses exist. The methods discussed above can be used, although hybridization and washing are performed under non-stringent conditions.

The DIMA of the present invention can also be used to obtain expression of LAV viral antigens for diagnostic purposes as well as to prepare a vaccine against LAV. Fragments particularly advantageous for this purpose will be described below.

The methods that can be used are diverse: a) DNA can be transfected into mammalian cells with appropriate selection markers using a variety of techniques, calcium phosphate precipitation, polyethylene glycol, protoplast fusion, etc.

b) DNA fragments corresponding to genes can be cloned into expression vectors for E. coli, yeast or mammalian cells and the resulting proteins can be purified.

C) The proviral DNA can be "shot-gunned" (fragmented) in prokaryotic expression vectors to generate fusion polypeptides. Recombinants producing antigenically competent fusion proteins can be identified simply by screening the recombinants with antibodies against LAVel1 or 20 IAVMAL antigens.

In this context, reference is made in particular to those parts of LAVEU and LAVMAl's genomes, which are shown in the figures as belonging to open reading frames, which encode the products having the polypeptide base structures shown.

25

More specifically, the present invention relates to the various polypeptides shown in FIG. 7A-8I. The methods described in EP 0 178 978 and PCT / EP85 / 00548 filed October 18, 1985 can be used to prepare such peptides from the corresponding viruses.

30

The present invention further aims to provide polypeptides containing common sequences with polypeptides comprising antigenic determinants contained in the proteins encoded and expressed by the LAVEU and LAVmal genome. It is a further object of the invention to further provide ways of detecting proteins related to these LAV viruses, in particular for diagnosis of AIDS or pre-AIDS or otherwise, for detecting antibodies against LAV virus or related proteins, especially in patients suffering from AIDS or in pre-AIDS, or more generally in asymptomatic carriers and in blood-related products. Finally, the present invention also aims to provide 5 immunogenic polypeptides and in particular protective polypeptides for use in the preparation of vaccines for AIDS or related syndromes.

IN,

The invention also relates to lower molecular weight polypeptide fragments having peptide sequences or fragments in common with those shown in FIG. 7A-8I.

10 smaller size fragments can be obtained using known techniques. For example, such a method comprises splitting the original major polypeptide with enzymes capable of cleaving it at specific sites. Examples of such proteins include the Staphylococcus aureus-V8 enzyme, c etc.

Other aspects of the present invention will be apparent from the following description of the data obtained from LAVeu and LAVMA1, with reference to the drawing, in which: 1A and 1B show restriction maps of LAVELi and LAVmal's genomes in comparison to LAVBru (a known LAV isolate deposited in CIMCM with the number 1-232 on July 15, 1983); FIG. 2 shows comparison charts showing the relative positions of the open reading frames of the genomes mentioned above; FIG. 3A-3F (hereinafter sometimes also referred to as Fig. 3) show the relative consistency of the proteins (or glycoproteins) 1 encoded by the open reading frames, with amino acid residues in the LAVELj and LAVMAL protein sequences indicated on a vertical line together with corresponding amino acid residues (numbered) in corresponding or homologous proteins or glycoproteins from LAVBru; 35 DK 175819 B1 6 fig. 4A-4B (hereinafter sometimes also referred to as Fig. 4) show a quantification of the sequence divergence between homologous LAVBRU, LAVEir and LAVmal proteins; FIG. 5 shows a diagram of the degree of divergence between the different virus envelope proteins; FIG. 6A and 6B (or Fig. 6, when considered together) show the direct repeats appearing in the proteins of the various AIDS virus isolates; and FIG. 7A-7J and 8A-8I show the complete nucleotide sequence of LAVEU and LAV template, respectively.

RESULTS

15 Characterization and molecular cloning of two African isolates

The various AIDS virus isolates in question are denoted by three letters from the patient's name, with LAVbru referring to the AIDS virus prototype, isolated in 1983 from a French gay patient with LAS, which was thought to have been infected in the United States in previous years ( Barré-Sinoussi et al., 1983). Both of the African patients originated from Zaire; LAVELI was isolated in 1983 from a 24-year-old woman with AIDS and LAVMA1 in 1985 from a 7-year-old boy with ARC, presumably infected in 1981 after a blood transfusion in Zaire, since his parents were LAV seronegative.

25

Isolation and purification of each of the two viruses was carried out by the procedure described in EP 138 667 filed September 9, 1984.

t-AVaj and LAVmal, by their structural and biological properties in vitro, are indistinguishable from the previously characterized isolates. Virus metabolic labeling and immunoprecipitation of patients' ELI and MAL sera as well as reference sera showed that the LAVFU and tAVMAL proteins had the same molecular weight (MW) and cross-reacted immunologically with prototype AIDS virus proteins (data not shown) of "LAV- 1 "class.

35 7 DK 175819 B1

Reference is again made to EP 178 978 and PCT / EP85 / 00548 regarding the purification, mapping and sequencing procedures used herein. See also "Experimental Procedures" and "Explanation of Figures" below.

Primary restriction enzyme analysis of LAVEU and LAVMAL genomes was performed by Southern blot with total DNA from acutely infected lymphocytes using - cloned complete LAVBRir genome as probe. Total cross hybridization was observed under stringent conditions, but the restriction profiles of the Zaire isolates were clearly different. Phage λ clones with the complete viral genetic information 10 were obtained and further characterized by restriction mapping and nucleotide sequence analysis; the E-H12 clone originates from LAVEU-infected cells and contains an integrated provirus with 5 'flanking cellular sequences but a truncated 3' long terminal repeat (LTR); The clone M-H11 was obtained by complete HindIII restriction of DNA from LAVMAi-infected cells, utilizing the existence of a unique HindIII site in LTR. Thus, M-H li is probably derived from non-integrated viral DNA, as this species occurred at least 10 times more frequently than integrated provirus.

FIG. IB shows a comparison between the LAVEU, LAV template, and LAVbru restriction maps, all three derived from their nucleotide sequences, and between 20 three Zaire isolates previously mapped for 7 restriction enzymes (Benn et al., 1985). Despite this limited number, all profiles are clearly different (out of the 23 sites that make up the LAVBRu map, only 7 are present in all the 6 maps shown), confirming the genetic polymorphism of the AIDS virus. There is no obvious relationship between the 5 cards from Zaire, and 25 of all their common places are also found in LAVBRU.

Conservation of the genetic organization

The genetic organization of LAVeu and LAVmal, as deduced from the complete nucleotide sequences of their cloned genomes, is identical to that found in other isolates, viz. 5'gag-pole-central region-env-F3 '. Most notable is the preservation of the "central region" (Fig. 2) located between the pole and env genes, which consists of a series of overlapping open reading frames (orf), previously referred to as Q, R, S , T and U, with a similar organization being observed in 35 sheep virus lentiviruses (Sonigo et al., 1985). the orf S product (also DK 175819 B1 8 designated "tat") is implicated in the transactivation of virus expression (Sodroski et al., 1985; Arya et al., 1985); the biological role of the orf Q product (also referred to as "sor" or orf A) is still unknown (Lee et al., 1986; Kang et al., 1986). Of the three other orphs (R, T, and U), only the orf R is likely to be a 7th viral gene for the following reasons: the exact preservation of its relative position with respect to Q and S (Fig. 2); the constant presence of a possible splicing acceptor and of a consensus AUG initiation codon, its corresponding codon use with respect to viral genes, and finally the fact that the variation in its protein sequence among the various isolates can be compared with the variation of JO among gag, pole and Q (see Fig. 4).

The sizes of the U3, R and U5 elements of the LTR are also preserved (data not shown) as are the location and sequence of their regulatory elements such as TATA box and AATAAA polyadenylation signal as well as their flanking sequences, i.e. primer binding site (PBS) complementary to the 3 'end of tRNA3LYS and polypurine region (PPT). The majority of genetic variability within the LTR is found in the 5 'half of U3 (encoding a portion of orf F), while the 3' end of U3 and R, which carry most of the cis-acting regulatory elements : promoter, enhancer and transactivating factor receptor (Rosen et al., 1985) as well as the U5 element are well preserved.

In general, it is clear that the isolates from Zaire belong to the same type of retrovirus as the previously sequenced isolates of American or European origin.

Variability of the viral proteins

Despite their identical genetic organization, these isolates exhibit significant differences in the primary structure of their proteins. The amino acid sequence of the LAVEU and LAVMAL proteins is shown in FIG. 3A-3F (to be considered together with Figures 7A-73 30 and 8A-8I) in line with the sequences of LAVBRU and ARV 2. Their divergence was quantified as the percentage of amino acid substitutions in 2-and-2 groupings (Fig. 4). . The number of insertions and deletions that may be introduced in each of these groupings is also listed.

9 DK 175819 B1

Three general observations can be made. First, the protein sequences of the African isolates are more divergent from LAVBRU than the sequences of HTLV-3 and ARV 2 (Fig. 4A); similar results are obtained if ARV 2 is used as a reference (not shown). The prevalence of genetic polymorphism between the isolates of the AlDS virus is 5 significantly greater than previously observed. Second, the two sequences in question confirm that the mantle is more variable than the gag and pole genes. The relatively small difference seen between env from LAVBRU and HTLV-3 again appears here as an exception. Third, the divergence between the two African isolates (Fig. 4B) is comparable to the divergence between LAVBWJ and each of them; 10 as far as can be extrapolated from only three sequenced isolates from the United States and

Europe and two from Africa, this indicates a more widespread evolution of the AIDS virus in Africa.

oaa oo pol: Their greater degree of conservation compared to the mantle corresponds to the fact that they encode important structural or enzymatic activities.

Among the three mature gag proteins, p25, which was the first recognized immunogenic LAV protein (Barré-Sinoussi et al., 1983), is also the best conserved (Fig. 3). In gag and pole, differences between isolates are primarily due to point mutations, and only a small number of insertion or detection events have been observed. Among these, 20 are noted the presence of a 12 amino acid (AA) insertion in the LAVBRU overlapping gag and pole, which is encoded by the No. 2 copy of a 36 bp direct repeat present only in this isolate and in HTLV-3. This duplication was omitted due to a computer error in the published LAVBru sequence (position 1712, Wain-Hobson et al., 1985), but was actually present in the HTLV-3 sequences (Ratner et al., 1985; Muesing et al., 1985).

env: In the envelope glycoprotein precursor, three segments can be distinguished (Allan et al., 1985; Montagnier et al., 1985; DiMarzoVeronese et al., 1985). The first is the signal peptide (position 1-33 in Fig. 3) and its sequence appears to be variable; The second segment (positions 34-530) constitutes the outer membrane protein (OMP or gp110) and carries most of the genetic variations, and in particular almost all of the numerous reciprocal insertions and deletions; the third segment (531-877) is separated from the OMP by a potential cleavage site following a constant base stretch (Arg-Glu-Lys-Arg) and forms the transmembrane protein 10 DK 175819 B1 (TMP or gp41) responsible for the anchoring of the envelope glycoprotein in the cell membrane. Better preservation of TMP than of OMP has also been observed between different mouse leukemia viruses (MLV, Koch et al., 1983) and may be due to structural bands.

5

From the grouping of FIG. 3 and the graphical indication of the sheath variability shown in FIG. 5, the existence of conserved domains with little or no genetic variation and hypervariable domains in which even the grouping of the different sequences is very difficult due to the existence of a large number of mutations and reciprocal insertions and deletions is evident. The sheath sequence of the HTLV-3 isolate is not included as it is so close to LAVBRU's (cf. Fig. 4), even in the hypervariable domains, that it adds nothing to the analysis. While this graphic designation is complemented by multiple sequence data, the general profile is already clearly visible with three hypervariable domains {Hyl, 2 and 3), all located in OMP and 15 separated by three well-preserved stretches (residues 37-130, 211-289 and 488-530 in the Fig. 3 grouping), presumably associated with important biological functions. Despite the immense genetic variability, the folding pattern of the envelope glycoprotein is likely constant. Namely, the location of all cysteine residues is conserved in the various isolates (Figs. 3 and 5), and the only three 20 variable cysteine residues are found either in the signal peptide or in the C-terminal part of the TMP. OMP's hypervariable domains are surrounded by conserved cysteine residues, suggesting that they may represent loops bound to the common folding pattern. The calculated hydropathic profiles (Kyte and Doo-little, 1982) of the various envelope proteins have also been remarkably preserved (not shown).

About half of the potential N-glycosylation sites, Asn-X-Ser / Thr found in the envelopes of the Zaire isolates, are mapped to the same positions in LAVBru (17/26 for LAVa, and 17/28 for LAVMAi). The other sites appear to be within 30 variable domains of env, suggesting the existence of differences in the degree of envelope glycosylation in different isolates.

Other viral proteins: Among the three other identified viral proteins, p27 encoded by orf F, 3 'of env (Allan et al., 1985b) is the most variable (Fig. 4). The 35 proteins encoded by orf Q and S from the central region are notable for their lack of insertions / deletions. A high frequency of amino acid substitutions comparable to that observed in env was surprisingly found for the orf S product (transactivating factor). On the other hand, the protein encoded by orf Q is no more variable than gag. The lower 5 variation of the proteins encoded by the central regions of LAVEU and LAVmal is also noteworthy.

DISCUSSION

With the availability of the complete nucleotide sequence from 5 independent isolators, some general features of the genetic variability of the AIDS virus are now emerging. First, the main reason for this is point mutations that very often result in amino acid substitutions and which are more frequent in the 3 'portion of the genome (orf S, env and orf F). Like all RNA viruses, retroviruses are thought to be highly susceptible to mutations due to RNA polymerase defects during their replication, with no proofreading at this stage (Holland et al., 1982; Steinhauer and In Holland, 1986).

Insertions / deletions are another source of genetic diversity. From FIG. In the 3-20 groupings, insertion events appear to be involved in most cases, as deletions would otherwise have occurred in independent isolates at the exact same site. Furthermore, after analyzing these insertions, it has been observed that they most often represent one of the two copies of a direct repeat (Fig. 6). Some are preserved perfectly like the 36 bp repeat in the 25 gag pole overlap of LAVBRU (Fig. 6-a); others carry point mutations leading to amino acid substitutions and, as a consequence, they are more difficult to detect, although clearly present, in the envervariable domains of the env (cf. Figures 6-g and 6-h). As noted for point mutations, the env gene and orf F also appear to be more susceptible to this kind of genetic variation than the rest of the 30 genome. The retention rate of these repeats must be associated with the time of their emergence in the analyzed sequences: the more degenerate, the older.

A very recent divergence between LAVBRU and HTLV3 is suggested by an extremely low number of mismatched AAs between their homologous proteins. However, one of LAVBRU's repeats (located in env's Hyl domain, Figs. 6-f) is not present in HTLV3, which indicates that this generation of tandem repeats is a rapid source of genetic diversity. . No trace of such a phenomenon has been found, even when very closely related viruses such as Mason-Pfizer monkey virus, MPMV (Sonigo et al., 1986) and an immunosuppressive simian virus SRV-1 (Power et al., 1986) has been compared. Insertion or deletion of one copy of a direct repeat has occasionally been described in mutated retroviruses (Shimotohno and Temin, 1981; Darlix, 1986), but the extent to which this phenomenon is now observed is unprecedented.

The molecular basis for these duplications is not clear, but could be the "copy-choice" phenomenon, due to the diploidity of the retroviral genome (Varmus and 10 Swanstrom, 1984; Clark and Mak, 1983). During the synthesis of the first strand of the viral DNA, it is known that leaps from one RNA molecule to another occur, especially when a fragment or stable secondary structure is present on the template; an inaccurate reinitialization on the second RNA template could result in the formation (or elimination) of a short direct repeat.

15

Genetic variability and subsequent antigenic modifications have often been developed by microorganisms as a means of escaping the host's immune response, either by modifying their epitopes during the course of infection, as is the case in trypanosomes (Borst and Cross, 1982), or by developing a large selection of antigens, as observed in influenza virus (Webster et al., 1982).

Since the human AIDS virus is related to animal lentiviruses (Sonigo et al., 1985; Chiu et al., 1985), its genetic variability could be a source of antigenic variation, which can be observed during the course of the infection with sheep lentivirus virus. (Scott et al., 1979; Clements et al., 1980) or with the infectious equine anemia virus (EIAV, Montelaro et al., 1984). However, a major deviation in these animal models is the extremely low or no neutralizing activity in the sera of individuals infected with AIDS virus, whether they are healthy carriers exhibiting minor symptoms or suffering from AIDS (Weiss et al., 1985; Clavel et al., 1985). Furthermore, the exact role of antigen variation in the pathogenesis, even for visna viruses, is unclear (Thormar et al., 1983; Lutley et al., 1983). Rather, it is believed that genetic variation represents a general selective advantage for lentiviruses by allowing adaptation to different environments, for example, by modifying their tissues or host tropisms. In the special cases of the AIDS virus, rapid genetic variations are tolerated, especially in the mantle; they could allow the virus to adapt to different "micro-environments" in the membrane of their main target cells, namely T4-! 13 DK 175819 B1, the lymphocytes. These "rhino chromosomes" could occur because the virus receptor is in the immediate vicinity of polymorphic surface proteins that differ either between individuals or between lymphocyte clones.

(5 Preserved domains in the AIDS virus envelope

As the proteins of most isolates are antigenically cross-reactive, the genotypic differences do not appear to affect the sensitivity of actual diagnostic tests based on detection of antibodies to AIDS virus and using purified 10 virions as antigens. Nevertheless, they must be taken into account in the development of the "second-generation" tests that are expected to be more specific, using less synthetic or genetically engineered viral antigens, the identification of conserved domains in the highly immunogenic envelope glycoprotein and also the structural nuclear proteins (gag) are very important in these tests, and the conserved stretch found at the end of OMP and at the beginning of TMP (490-620, Fig. 3) could be a good candidate, as a bacterial fusion protein containing this domain was well detected by the sera of AIDS patients (Chang et al., 1985).

The envelope, specifically OMP, mediates the interaction between a retrovirus and its specific cellular receptor (DeLarco and Todaro, 1976; Robinson et al., 1980). In the case of AIDS virus, in vitro binding assays have shown that the envelope glycoprotein gp110 interacts with the T4 cell surface antigen (McDougal et al., 1986), which is already thought to be the virus receptor or closely related to it (Klatzmann 25 et al., 1984; Dalgleish et al., 1984). Identification of the AIDS virus envelope domains responsible for this interaction (receptor binding domains) seems to be fundamental to understanding the host-virus interactions, but also to the design of a protective vaccine, as an immune response to these epitopes could presumably trigger neutralizing antibodies. . Since the AIDS-30 virus receptor is at least partially constituted by a constant structure, the T4 anti-gene, the envelope binding site is probably not encoded solely by domains undergoing drastic genetic alterations between isolates, even if implicated in one or the other. kind of an "adaptation". One or more of the conserved domains in the OMP (residues 37-130, 211-289 and 488-530 in Fig. 3-35 grouping), which are brought together by the folding of the protein, may play a role in the interaction. between virus and receptor, and this can be explored with synthetic or genetically engineered peptides derived from these domains, either by direct binding assays or indirectly by examining the neutralizing activity of specific antibodies formed against them.

: 5

African AIDS viruses

Zaire and the neighboring countries of Central Africa are considered an endemic area for AIDS virus infection and the possibility of the virus originating in Africa has become the subject of intense exchange of views (see Norman, 1985). Based on the present study, it is clear that the genetic organization of isolates from Zaire is the same as that of US isolates, indicating a common origin.

The very significant sequence differences observed between the proteins are consistent with a divergent evolutionary process. In addition, the two African-15 isolates among themselves are more divergent than the US isolates already analyzed; insofar as the observation can be extrapolated, it suggests a longer evolution of the virus in Africa, and this is also consistent with a greater proportion of the population being exposed than in developed countries.

A novel human retrovirus with morphology and biological properties (cytopathogenicity ·, T4 tropism), similar to LAV's, but nonetheless distinctly genetically and antigenically distinct from the latter, was recently isolated from two Guinea Bissau AIDS patients, West Africa (Clavel et al., 1986). In neighboring Senegal, the population appears to be exposed to a retrovirus that is also different from LAV but which is apparently not pathogenic (Barin et al., 1985; Kanki et al., 1986). Both of these novel African retroviruses appear to be antigenically related to simian T-cell lymphotrophic virus, STLV-III, which has been shown to be widespread in healthy African green monkeys and other simian species (Kanki et al., 1985). This allows for a large group of African primate lentiviruses extending from the apparently apatogenic simian viruses to LAV-type viruses.

Their precise interrelationships will only be known once they are fully characterized in genetic terms, but they are already very likely to have originated from a common ancestor. The considerable genetic variability observed between AlDS virus isolates in Central Africa is probably a characteristic of this entire group and may be responsible for the apparently significant genetic divergence between its members (loss of cross-antigenicity in the cloak). In this regard, the preservation of tropism for T4 lymphocytes suggests that these retroviruses have a major advantage.

5 EXPERIMENTAL PROCEDURES

Isolation of viruses LAVeu and LAVmal were isolated from patients' peripheral blood lymphocytes as described (Barré-Sinoussi et al., 1983); Briefly, the lymphocytes were fractionated and co-cultured with phytohemagglutinin-stimulated normal human lymphocytes in the presence of interleukin 2 and anti-α interferon serum. Virus production was measured by performing cell-free reverse transcriptase (RT) activity assay in the cultures and by electron microscopy.

15

Molecular cloning

Normal donor lymphocytes were acutely infected (104 cpm RT activity / 106 cells) as described (Barré-Sinoussi et al., 1983), and total DNA was extracted at the beginning of the RT activity peak. For LAVEU, an λ library was constructed using the L47-1 vector (Loenen and Brammar, 1982) by partial HindIII cutting of the DNA as described previously (Alizon et al., 1984). For LAVMA1, DNA from infected cells was completely cut with HindIII and the 9-10 kb fraction was selected on 0.8% low melting agarose gel and ligated into L47-1 HindIII arms. Ca. 5x10 7 '/ 45 25 plaques for LAVEL] and 2x10745 for LAV template obtained by in-vitro packaging (Amersham) were plated on E. coli LA101 and screened in situ under stringent conditions using as the probe the 9 kb Sacl insertion from the clone λ 319 (Alizon et al., 1984) carrying the majority of the LAVBRu genome. Clones exhibiting positive signals were plaque purified and propagated on E. coli C600 30 recBC, and two recombinant phages carrying the complete genetic information for LAVel (E-H12) and LAVmal (M-Hll) were further characterized by restriction mapping.

Nucleotide Sequence Strategy 35 16 DK 175819 B1

Viral fragments derived from E-H12 and M-Hll were sequenced by the dideoxy chain termination procedure (Sanger et al., 1977) following "shotgun," cloning in the M13mp8 vector (Messing and Viera, 1982), as previously described (Sonigo et al., 1985). The LAVEU viral genome is 9176 nucleotides long and that of I5 viral IAVMAL genome 9229 nucleotides long. Each nucleotide was determined from more than 5 independent clones on average. Full nucleotide sequences are not disclosed here for obvious space reasons, but are freely available upon request from the authors until released via sequence data banks.

10 FIGURE EXPLANATION

FIG. 1: Restriction map analysis of AIDS virus isolates A) Restriction map of insertions into phage λ clones from cells infected with LAVCU (E-15 H12) and LAVmal (M-Hll). The schematic genetic organization of the AIDS virus is listed above the maps. The LTRs are marked with filled boxes. A: AvaI, B: BamHI, Bg: BglII, EiEcoRI, H: HindIII, Hc: HincIl, KrKpnl, N: NdeI, P: PstI, S: SacI, X: XbaI. Asterisks indicate the HindIII cloning sites in the λ L47-1 vector.

20 B) Comparison of the sites of 7 restriction enzymes in 6 isolates: prototype AI DS virus LAVBRU, LAVmal and LAVFL); Z1, Z2, Z3 are Zaire isolates with published restriction maps (Benn et al., 1985).

Restriction sites are indicated by the following symbols: Bglll; Eco;

Hindi; HindIII; Kpn I; Nde; Sac.

25

FIG. 2: Conservation of the genetic organization of the central region in AIDS virus isolates

Stop codons in each phase are shown as vertical lines. Vertical arrows indicate possible 30 AUG initiation codons. Splice acceptor (A) and donor sites (D) identified in sub-genome viral mRNA (Muesing et al., 1985) are shown below the drawing for LAV use and corresponding sites in LAVEU and LAV template. PPT indicates the repeat of the polypurine region flanking the 3'LTR. As observed in LAVBRU (Wain-Hobson et al., 1985), PPT 256 nucleotides are repeated 5 'relative to the end of the pol gene in both of the sequences, but this repeat is degenerate in two positions in LAV ELI

J

FIG. 3: Grouping of the protein sequences of 4 AIDS virus isolates

The LAVBRU isolate (Wain-Hobson et al., 1985) is used for reference; differences relative to LAVBRu are noted only for ARV2 (Sanchez-Pescador et al., 1985) and the two isolates of Zaire LAVMAi and LAVEU. A minimum number of gaps (-) was introduced into the groupings. The NH2 terminals for p259a9 and pl8ga9 are indicated (Sanchez-Pescador, 1985). The potential cleavage sites in the mantle precursor (Allan et al., 1985a; diMarzo-Veronese, 1985) separating the signal peptide (SP), the outer membrane protein (OMP) and the transmembrane protein (TMP) are indicated as vertical arrows; conserved cysteine residues are indicated by black circles, and variable 15 cysteine residues are surrounded by a box. The one-letter code for the amino acids is: A: Ala, C: Cys, D: Asp, E: G! U, F: Phe, G: Gly, H: His, small, K: l_ys, L: Leu, M: Met, N : Asn, P: Pro, Q: Gln, R: Arg, S: Ser, T: Thr, V: Val, W: Trp, Y: Tyr.

FIG. 4: Quantification of the sequence divergence between homologous proteins from 20 different isolates

Part A of each table lists results derived from two-by-two groupings using the LAVBRu proteins as reference, and Part B the results where the LAVeu proteins are used as reference. Sources: Muesing et al., 1985 for HTLV-3; Sanchez-Pescador et al., 1985 for ARV 2 and Wain-Hobson et al., 1985 for LAVbru. In each of the tables, the size of the protein in amino acids (calculated from the first methionine residue or for pole from the beginning of orf) is given in the upper left. The number of deletions (left) and insertions (right) required for the grouping is given below. The large highlighted figures indicate 30 percent of amino acid substitutions (insertions / deletions excluded). Two-and-two groupings were performed using computer (Wilburg and Lipman, 1983) using a gag penalty of 1, K-tuple of 1, and window of 20 with the exception of env's hypervariable domains, where the number of gaps was made as small as possible and which are mainly grouped as shown in Figs. 3. The sequence of the expected protein encoded by HTLV-3 orf R has not been compared due to premature termination relative to all other isolates.

Fig. 5: Variability of the AIDS virus envelope protein at 5 At each position x in the grouping of env (Fig. 3), variability V (x) was calculated as the number of different amino acids at position x 10 V (x) = the frequency of the most frequently occurring amino acid. in position

Gaps in the groupings are considered another amino acid. In a group of 4 proteins, V (x) extends from 1 (identical AA in the 4 sequences) to 16 (4 different 15 AA). This form of preparation has been used previously in a compilation of the AA sequence into the variable regions of immunoglobulins (Wu and Kabat, 1970). Vertical arrows indicate the cleavage sites; asterisks indicate potential N-glycosylation sites (N-X-S / T) conserved in all 4 isolates; black triangles indicate conserved cysteine residues. Black rhombs mark the three major hydrophobic domains. OMP: 20 outer membrane protein; TMP: transmembrane protein; signal: signal peptide; Shelf, 2, 3: hypervariable domains.

FIG. 6: Direct repeats in the proteins of various AIDS virus isolates 25 These examples are derived from the ones in FIG. 3 grouped sequences of gag (a, b), F (c, d) and env (e, f, g, h). The direct repeats two elements are surrounded by a box, while degenerate positions are emphasized.

Thus, the invention relates more specifically to the proteins, polypeptides or glycoproteins, including the polypeptide structures shown in the drawings. The first and last amino acid residues of these proteins, polypeptides or glycoproteins are indicated by numbers calculated from a first amino acid in the relevant open reading frames, although these numbers do not exactly match the numbers of the LAV (or LAVMAL) proteins concerned. but rather to the numbers of the corresponding LAVBru proteins or sequences shown in FIG. 3A, 3B and 3C. Thus, a number corresponding to a "first amino acid residue" in a LAVEU protein corresponds to the number of the first amino acid residue in the corresponding LAVBRu protein, which in each of Figures 3A, 3B or 3C is in direct grouping with the corresponding first amino acid of the LAVELr protein.

The relevant sequences can thus be read from FIG. 7A-7J and 8A-81 to the extent that they are not sufficiently clear in FIG. 3A-3F.

The preferred protein sequences of the present invention range from the corresponding "first" and "last" amino acid residues (also referred to the protein or glycoprotein moieties comprising portions of the following sequences): 10 OMP or gp110 proteins comprising precursors: 1 to 530 OMP or gp110 without precursor: 34-530 Sequence bearing TMP or gp41 protein: 531-877, especially 1 680-700

Well-preserved OMP areas: 37-130 I 20 211-289 and 488-530

Well-preserved area found at the end of the OMP and at the beginning of the TMP: 490-620.

25

Proteins containing or consisting of the "well-conserved regions" are of particular interest in the preparation of immunogenic preparations and (preferably in relation to the regions of the env protein) of vaccine preparations against the class 1 LAV viruses defined above.

30

More specifically, the invention relates to all of the DNA fragments more specifically set forth in the drawings corresponding to open reading frames. It will be understood that those skilled in the art will be able to obtain all of them, for example, by cleavage of a complete DNA molecule corresponding to the complete genome of either LAVEU or LAVMA1, such as by cleavage by a partial or complete cutting thereof with a suitable restriction enzyme and by subsequent isolation of the relevant fragments. The various DNA molecules described above can also be used as a source of convenient fragments.

The techniques described in the PCT application for isolating the fragments which can then be inserted into appropriate plasmids can also be used here.

Other methods can of course be used. Some of these are exemplified in EP 178,978, filed September 17, 1985. For example, reference may be made to the following 10 methods: a) DNA can be transfected into mammalian cells with suitable selection markers using a variety of techniques, calcium phosphate seeding, polyethylene glycol, protoplast fusion, etc. .

B) DNA fragments corresponding to genes can be cloned into expression vectors for E. coli, yeast or mammalian cells, and the resulting proteins can be purified.

c) The proviral DNA can be shot-gunned (fragmented) into prokaryotic expression vectors to form fusion polypeptides. Recombinants producing antigenically competent fusion proteins can be identified by simply screening the recombinants with antibodies against LAV antigens.

The invention further relates more specifically to DNA recombinants, especially modified vectors comprising any of the foregoing DNA sequences, and adapted to transform corresponding microorganisms or cells, especially eukaryotic cells such as yeast, e.g., Saccharomyces cerevisiae, or higher eukaryotic cells, especially mammalian cells, and to enable expression of the DNA sequences in the corresponding microorganisms or cells. General procedures of this type are summarized in the above PCT application PCT / EP85 / -00548 filed October 30, 1985.

In particular, the invention relates to such modified DNA recombinant vectors that are modified by the aforementioned DNA sequences and which are capable of transforming higher eukaryotic cells, especially mammalian cells. Preferably, any of the above 35 sequences are placed under the direct control of a promoter contained in these vectors, which is recognized by the polymerases of the cells concerned, so that the first expressed nucleotide codons correspond to the first triplets in the DNA sequences defined above. Accordingly, the present invention also relates to the corresponding DNA fragments obtainable from the LAVeli or LAVMA1 genomes or similar cDNA molecules using any convenient method. Such a method comprises, for example, cleaving the LAV genomes or cDNA molecules with restriction enzymes, preferably at the level of restriction sites surrounding the fragments and close to their respective opposite ends, isolating and identifying the desired 10 fragments by size, their restriction maps or if necessary, nucleotide sequences are examined (or by reaction with monoclonal antibodies specifically directed to epitopes carried by the polypeptides encoded by the DNA fragments) and, if necessary, also trim the ends of the fragment, for example by means of an exonucleolytic enzyme such as Bal3l, for the purpose of controlling the desired nucleotide sequences at the ends of the DNA fragments or opposite, these ends being filled with Klenow enzyme, and optionally ligating them to synthetic polynucleotide fragments designed to allow reconstitution of the nucleotide fragments. These fragments can then be inserted into any of the vectors mentioned to effect expression of the corresponding polypeptide by the cell transformed therewith. The corresponding polypeptide can then be isolated from the transformed cells, if necessary after lysis thereof, and purified by methods such as electrophoresis. Of course, all traditional methods for performing these operations can be used.

25

The invention also relates more specifically to cloned probes that can be prepared from any DNA fragment of the present invention, and thus relates to recombinant DNA molecules containing such fragments, in particular any plasmid amplifiable in prokaryotic or eukaryotic cells. , and which carry the fragments.

Using the cloned DNA fragments as a molecular hybridization probe - either by labeling with radioactive nucleotides or fluorescent reagents - LAV virion RNA can be detected directly in the blood, body fluids and blood products (e.g., by the anti-hemolytic factors such as Factor VIII concentrates) and vaccines; hepatitis B vaccine. It has already been demonstrated that intact virus can be detected in culture supernatants from LAV-producing cells. A convenient method of obtaining this detection involves virus being immobilized on a substrate, e.g., nitrocellulose filters, etc., destroying the virion, and by hybridizing with labeled (radiolabelled or "cold" fluorescence or enzyme labeled) probes. Such a method has already been developed for peripheral blood hepatitis B virus (according to J. Scotto et al., Hepatology (1983), 3, 379-384).

Probes of the present invention can also be used for rapid screening of 10 genomic DNA from tissues from patients with LAV-related symptoms to see if the proviral DNA or RNA present in the host tissue and other tissues can be related that from LAVEU or LAVmae.

A method that can be used for such screening comprises the following 15 steps: extraction of DNA from tissue, restriction enzyme cutting of the DNA, electrophoresis of the fragments, and Southern blotting of genomic DNA from tissue, subsequent hybridization with labeled cloned LAV. proviral DNA. In situ hybridization can also be used.

20 Lymphatic fluids and tissues and other non-lymphatic tissues from humans, primates and other mammalian species can also be screened to see if other evolutionarily related retroviruses exist. The methods discussed above can be used, although hybridization and washing are performed under non-stringent conditions.

The DNA molecules or DNA fragments of the present invention may also be used to obtain expression of viral antigens from LAVEU or LAV template for diagnostic purposes.

The invention generally relates to the polypeptides themselves, whether chemically synthesized, isolated from viral preparations, or expressed by the various DNA molecules of the invention, especially the ores or fragments thereof, in suitable hosts, especially prokaryotic or eukaryotic hosts. after these have been transformed with a suitable vector which has been previously modified with the corresponding DNA molecules. More generally, the invention also relates to any of the polypeptide fragments 35 (or molecules, especially glycoproteins having the same polypeptide base structure as the above-mentioned polypeptides), which carry an epitope characteristic of a protein or glycoprotein of LAVeu. or LAVMA1, which polypeptide or molecule then has N-terminal and C-terminal ends, respectively, that are either free or independently covalently linked to amino acids different from those normally associated with those in the larger polypeptides or glycoproteins of the LAV virus, which latter amino acids are thus free or belong to another polypeptide sequence5. In particular, the invention relates to hybrid polypeptides containing any of the epitope-bearing polypeptides defined more specifically above, recombined with other polypeptide fragments that are normally foreign to the LAV proteins and of sufficient size to provide increased immunogenicity of the epitope-bearing polypeptide, however, the foreign polypeptide fragments are either immunogenically inert or do not interfere with the immunogenic properties of the epitope-bearing polypeptide.

Such hybrid polypeptides, which may contain from 5 up to 150, even 250 amino acids, usually consist of the expression products of a vector which initially contained a nucleic acid sequence which can be expressed under the control of a suitable promoter or replicon in a suitable host. however, which nucleic acid sequence had been modified in advance by inserting therein a DNA sequence encoding the epitope-bearing polypeptide.

These epitope-bearing polypeptides, especially those whose N-terminal and C-terminal amino acids are free, can also be obtained by chemical synthesis according to techniques well known in the protein chemistry.

25

Synthesis of peptides in homogeneous solution and in solid phase is well known.

In this regard, the homogeneous solution synthesis method described by Houben-Weyl can be used in the work entitled "The Method of Organizational Chemistry" edited by E. Wunsch, Vols 15-1 and II, Thieme, Stuttgart, 1974.

This method of synthesis consists in successively condensing the successive amino acids two and two, or successively condensing already available or forming successive peptide fragments already containing several 35 aminoacyl residues in the correct order. With the exception of the carboxyl and amino groups involved in the formation of the peptide bonds, care must be taken that all other reactive groups carried by these amino acid groups or fragments are protected in advance. However, prior to the formation of the peptide bonds, the carboxyl groups are advantageously activated using methods well known in the peptide synthesis. Alternatively, coupling reactions can be used using conventional coupling reagents, for example the carbodiimide type such as 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide. When the amino acid group used carries an additional amino group (e.g., lysine) or another acid function (e.g., glutamic acid), these groups may be protected by carbobenzoxy or tert-butyloxycarbonyl groups for the amine groups or with tert-butyl ester groups for the carboxyl groups. . Similar methods are available to protect other reactive groups. For example, the SH group (e.g. in cysteine) may be protected with an acetamidomethyl or paramethoxybenzyl group.

In the case of progressive synthesis, amino acid by amino acid, the synthesis preferably starts by condensing the C-terminal amino acid with the amino acid corresponding to the neighbor amino acid group in the desired sequence, and then step by step until the N-terminal amino acid. Another preferred technique that can be used is that described by R.D. Merrifield in "Solid phase peptide synthesis" (J. Am.

Chem. Soc., 45, 2149-2154).

In accordance with the Merrifield method, the first C-terminal amino acid in the chain is fixed to a suitable porous polymer resin by its carboxyl group, after which the amino group of the amino acid is then protected, for example by a tert-butyloxycarbonyl group.

Thus, when the first C-terminal amino acid is fixed to the resin, the protecting group of the amine group is removed by washing the resin with an acid, ie.

Trifluoroacetic acid when the amine protecting group is a tert-butyloxycarbonyl group.

Then, the carboxyl group of the second amino acid to provide the second aminoacyl group of the desired peptide sequence is coupled to the deprotected amine group of the C-terminal amino acid fixed to the resin. The carboxyl group 25 of this second amino acid has preferably been activated, for example with dicyclohexylcarbo diimide, while its amine group has been protected, for example with a tert-butyloxycarbonyl group. In this way, the first portion of the desired peptide chain comprising the first two amino acids is obtained. As before, the amine group is then deprotected, and one can continue to fix the next aminoacyl group and so on until all of the desired peptide is obtained.

The protecting groups for the possibly different side groups of the peptide chain thus formed can then be removed. The desired peptide can then be released from the resin for example by hydrofluoric acid and finally isolated in pure form from the acid solution according to conventional procedures.

As for the smallest size peptide sequences, which sequences carry an epitope or immunogenic determinant, and especially those sequences readily available by chemical synthesis, it may be necessary to covalently or "conjugate" them to a physiologically acceptable and not -toxic carrier molecule to increase their immunogenic nature in vivo.

Examples of carrier molecules or macromolecular substrates which can be used to prepare the conjugates of the present invention include natural proteins such as tetanus toxoid, ovalbumin, serum albumin, hemocyanins, etc. Synthetic macromolecular carriers, e.g., polylysines or poly (DL). alanine) poly (L-lysine) 's may also be used.

Other types of macromolecular carriers which can be used and which generally have molecular weights above 20,000 are known from the literature.

The conjugates can be prepared by known methods as described by Frantz and Robertson in "Infect, and Immunity", 33, 193-198 (1981) or by P.E. Kauffman in 30 "Applied and Environmental Microbiology", October 1981, Volume 42, No. 4, 611-614.

For example, the following coupling agents may be used: glutaraldehyde, ethyl chloroformate, water-soluble carbodiimides (N-ethyl-N '- (3-dimethylamino-propyl) carbodiimide, HCl), diisocyanates, bis-diazobenzidine, di- and trichloro-s-triazines, cyanogen bromides, 26 DK 175819 B1 benzaquinone, as well as the coupling agents mentioned in "Scand. J. Immunol.", 1978, Vol. 8, pp. 7-23 (Avrameas, Ternynck, Guesdon).

Any coupling process can be used to bind one or more reactive groups in the peptide on the one hand and one or more reactive groups in the carrier on the other. Again, the coupling between carboxyl and amine groups carried by the peptide and the carrier is obtained, or conversely advantageously in the presence of a coupling agent of the type used in protein synthesis, i.e. 1-Ethyl-3- (3-dimethylaminopropyl) carbodiimide, N-hydroxybenzotriazole, etc. The coupling 10 between amine groups carried by the peptide and the carrier, respectively, can also be made with glutaraldehyde, for example by the method described by P. Boquet et al. (1982) Molec. Immunol., 19, 1441-1549 when the carrier is haemocyanin.

The immunogenicity of epitope-bearing peptides may also be enhanced by oligomerization thereof, for example in the presence of glutaraldehyde or any other suitable coupling agent. In particular, the invention relates to water-soluble immunogenic oligomers thus obtained, comprising in particular from 2 to 10 monomer units.

The glycoproteins, proteins and polypeptides (generally referred to herein as "antigens") of the invention, whether obtained (by methods such as those described in previous patent applications referred to above) in the purified state from LAVel] - or LAVMAL virus preparations or - especially with respect to the peptides - by chemical synthesis are useful in methods for detecting the presence of anti-LAV antibodies in biological media, especially biological fluids such as human or animal sera, especially for the purpose of possibly diagnosing LAS or AIDS.

In particular, the invention relates to an in vitro diagnostic method using a sheath glycoprotein (or a polypeptide carrying an epitope of this LAVClJ or LAVMA1 '30 glycoprotein) for the detection of anti-LAV antibodies in sera from subjects carrying them. Other polypeptides - especially those carrying an epitope of a nuclear protein - may also be used.

A preferred embodiment of the method according to the invention comprises that: a predetermined amount of one or more of the antigens is placed in the recesses of a microtiter plate; increasing dilutions of the biological fluid, viz. serum to be diagnosed is filled in these recesses; 5 - microplates are incubated; the microplate is carefully washed with a suitable buffer; specific labeled antibodies against blood immunoglobulins are added to the wells; and the antigen-antibody complex formed is detected, thus indicating the presence of LAV antibodies in the biological fluid.

The labeling of the anti-immunoglobulin antibodies is advantageously achieved with an enzyme selected from those capable of hydrolyzing a substrate, which substrate undergoes a modification in its radiation absorption, at least within a predetermined band of wavelengths. The detection of the substrate, preferably against a control, then provides a measure of the potential risks or the actual presence of the disease.

Thus, preferred methods are immunoenzymatic or immunofluorescent detections, especially according to the ELISA technique. Titrations may be immunofluorescence assays or direct or indirect immunoenzymatic assays. Quantitative antibody titrations on the sera examined can be performed.

The invention also relates to diagnostic kits per se for in vitro detection of LAV virus antibodies, which kits comprise any of the polypeptides identified herein and all the biological and chemical reagents and equipment necessary for carrying out of diagnostic assays. Preferred kits include all reagents required to perform ELISA assays. Thus, in addition to any of the aforementioned polypeptides, preferred 30 kits include suitable buffers and anti-human immunoglobulins, which anti-human immunoglobulins are labeled either with an immunofluorescent molecule or with an enzyme. In the latter case, preferred kits also comprise a substrate which can be hydrolyzed by the enzyme and which gives a signal, especially modified absorption of radiation, at least at a certain wavelength, which signal then indicates the presence. of an antibody in the biological fluid to be analyzed using the kit.

Of course, it may be advantageous to use multiple proteins or polypeptides not only from both LAVeLI and LAVMal, but also any or both of them together with homologous proteins or polypeptides of previously described viruses, e.g., LAVBru or HTLV-III or ARV, etc.

The invention also relates to vaccine preparations whose active ingredient is to consist of any of the antigens, i.e. the entire polypeptide antigens described above, from either LAVIIJ or LAVMai_ or both, especially the purified gp110 or immunogenic fragments thereof, fusion polypeptides or oligopeptides together with a suitable pharmaceutically or physiologically acceptable carrier.

A first type of preferred active ingredient is the gp110 immunogen of these immunogens.

Other preferred active substances to be considered in this field are those peptides containing less than 250 amino acid units, preferably less than 150, especially from 5 to 150 amino acid residues, as can be deduced for the complete LAV and LAVmai genomes, and especially those peptides containing one or more groups selected from Asn-X-Ser and Asn-X-Ser as defined above. Preferred peptides for use in the preparation of vaccines are peptides a) to f) as defined above. By way of example, which should not be considered as limiting, it may be mentioned that suitable doses of the vaccine preparations are those doses which can release antibodies in vivo in the host, especially a human host. Suitable doses range from 10 to 500 µg polypeptide, protein or glycoprotein per ml. kg, eg 50 to 100 pg per day. kg.

The various peptides of the present invention can also be used per se for the production of antibodies, preferably monoclonal antibodies specific to the respective various peptides. Conventional preparation and screening methods are used to prepare hybridomas secreting these monoclonal antibodies. These monoclonal antibodies, which are themselves part of I

The invention then provides a very useful tool for identifying and even determining relative amounts of the various polypeptides or proteins in biological samples, especially human samples containing LAV or related viruses.

The invention further relates to the hosts (prokaryotic or eukaryotic cells) which are transformed with the above recombinants and which are capable of expressing the DNA fragments.

Finally, the invention also relates to vectors for transformation of eukaryotic cells of human origin, especially lymphocytes whose polymerase is capable of recognizing 10 LAV's LTRs. The vectors are particularly peculiar to the presence of a LAV-LTR therein, which LTR is then active as a promoter, enabling in a suitable host efficient transcription and translation of a DNA insert encoding a particular protein placed under its control .

The invention, of course, relates to all variants of genomes and corresponding DNA fragments (ORFs) which have substantially equivalent properties, all of the genomes being retroviruses which may be considered equivalent to LAV.

It is to be understood that the following claims must also cover all equivalents of the products (glycoproteins, polypeptides, DNA molecules, etc.), the equivalent being a product, ie. a polypeptide which may differ from a particular product defined in any of the claims, for example in the form of one or more amino acids, while still having substantially the same immunological or immunogenic properties. A similar rule of equivalence should apply to the DNA molecules, since it is clear that the equivalence rule is then linked to the equivalence rule that applies to the polypeptides for which they encode.

It is also clear that all of the literature referred to above and below, and any patent applications or patents not specifically identified herein, but analogous to those specifically mentioned, should be considered to be mentioned herein by reference.

It is further to be noted that the invention further relates to immunogenic compositions which preferably do not contain only any of the polypeptides more specifically identified above and which have the amino acid sequences of LAVgu and LAVmai which have been identified, but also similar peptide sequences for previously defined LAV proteins.

In this regard, the present invention relates in particular to the particular polypeptides having the sequences which more specifically correspond to the LAVBRu sequences previously referred to, i.e. the sequences that extend between the following first and last amino acids of the LAVBRU proteins themselves, viz. those polypeptides having sequences contained in LAVbru-OMP or LAVbru-TMP, or sequences extending over both, especially those sequences spanning the following 10 positions of the amino acids contained in the env's open reading frame in the LAVBRL genome: 1-530 34-530 and preferably 531-877, especially 680-700 37-130 211-289 20 488-530 490-620.

These various sequences can be used for any of the purposes defined above and in any of the compositions described.

25

Finally, the invention also relates to the various antibodies that can be specifically produced against the various peptides described herein, especially the monoclonal antibodies that specifically recognize them. The corresponding hybridomas that can be formed from spleen cells that have been previously immunized with such peptides and fused with suitable myeloma cells and selected by standard procedures also form part of the invention.

The phage λ clone E-H12, which originates from LAVEU-infected cells, was deposited on May 9, 1986 in the Collection Nationale de Cultures de Microorganisms (CNCM) at Institut 35 Pasteur, Paris, France, with the number 1-550.

31 DK 175819 B1

The phage λ clone M-Hll, derived from LAVMAL-infected cells, was deposited in CNCM under May 1-551, under the number 1-551.

32 DK 175819 B1

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Claims (21)

42 DK 175819 B1 1. Purified HIV-1 variant virus, characterized in that it differs from LAVeRU in 9.8% or more of the amino acids of the Gag protein, in 5.5% or more of the amino acids of the Pol polypeptide and in 20, 7% or more of the amino acids in the full Env protein; by antibodies in AIDS patient serum binding to Gag, Pol or Env polypeptides from this HIV-1 variant virus; and in that the genetic structure of this HIV-1 variant is 5'-gag - pol - orf Q - ofr R - tat - orf U - env - orf F - 3 '. 10 The HIV-1 variant virus according to claim 1, which corresponds to the LAVEU virus whose RNA corresponds to the cDNA of FIG. 7A-7J. The HIV-1 variant virus according to claim 1, which corresponds to the 1_AVma1. virus whose RNA corresponds to the cDNA of FIG. 8A-8I. 4. The cDNA of FIG. 7A-7J. 5. The cDNA of FIG. 8A-8I. 20 5 OMP or gp110 proteins comprising precursors: 1 to 530 OMP or gp110 without precursor: 34-530 sequence carrying the TMP or gp41 protein: 10 531-877, especially 680-700 well-preserved OMP regions: 37-130 , 211-289 and 15 488-530 well-preserved area found at the end of the OMP and at the beginning of the TMP: 490-620. The DNA recombinant, characterized in that it contains the cDNA of claim 4 or 5. Probe, characterized in that it contains a cloned nucleic acid according to any one of claims 4 to 6. A method for identifying the presence in a host tissue of a virus or provirus related to either LAVeu or LAVMA1, characterized in that DNA obtained from the tissue is hybridized with a probe according to claim 7, and the presence of the virus or provirus in the tissue is detected. Protein or glycoprotein, characterized in that they are encoded by open reading frames in the DNA sequences of claims 4 or 5, or fragments thereof corresponding to regions ranging from amino acid group 37 to amino acid residue 130 or from amino-acyl group 211 to amino acid residue 289 or from amino acid residue 488 to amino-acyl group 530 in Figure 3. Part of a protein or glycoprotein according to claim 9, characterized in that it corresponds to the region extending from amino acid residue 490 to amino acid residue 620 in FIG. 3. 10 Part of a protein or glycoprotein according to claim 9, characterized in that its amino acid sequence comprises all or part of the following sequences: A method for in vitro detection of the presence of antibodies against LAVeu or LAVmal in human body fluids, characterized in that the body fluids are contacted with antigens obtained from the viruses of any one of claims 1 to 3, or consist of proteins, glycoproteins or portions thereof according to any one of claims 9 to 11, and that the immunological reaction between the antigens and antibodies is detected. The method of claim 12. 5 characterized in that a predetermined amount of one or more of the antigens is placed in the recesses of a microtiter plate; increasing dilutions of the biological fluid, viz. serum to be diagnosed is filled in these recesses; the microplate is incubated; the microplate is washed with a suitable buffer; specific labeled antibodies against blood immunoglobulins are added to the wells; and 15. the antigen-antibody complex formed is detected, which then indicates the presence of LAV antibodies in the biological fluid. Diagnostic kit for in vitro detection of antibodies against the viruses according to any one of claims 1 to 3, 20, characterized in that it contains an antigen specific to these viruses or consists of a protein, glycoprotein or part thereof according to any of claims 9-11, and the biological and chemical reagents as well as equipment necessary to perform diagnostic assays. An immunogenic composition, characterized in that it contains an antigen specific to the viruses of any one of claims 1 to 3, or any immunogenic protein, glycoprotein or part thereof of any one of claims 9-11 together. with a suitable pharmaceutically or physiologically acceptable carrier. 30 OMP or gp110 proteins comprising precursors: 1 to 530 OMP or gp110 without precursor: 34-530 sequence carrying the TMP or gp41 protein: 20 531-877, especially 680-700 well-preserved OMP regions: 37-130 , 211-289 and 25 488-530 well-preserved area found at the end of the OMP and at the beginning of the TMP: 490-620. Immunogenic preparation according to claim 15, characterized in that the protein, glycoprotein or part thereof is the gp110 envelope glycoprotein or part thereof. Immunogenic composition according to claim 16, characterized in that it contains a portion of a protein or glycoprotein according to claim 9, whose amino acid sequence comprises all or part of any of the following sequences: An antibody, especially a monoclonal antibody, characterized in that it is formed against any of the proteins, glycoproteins or portions thereof according to any one of claims 9-11. Cell, characterized in that it is transformed with a DNA recombinant according to claim 6. A method of producing DNA according to claim 4 or 5, comprising the steps of: 30. isolating the DNA of LAVeu or LAVMA1; or • that the DNA of LAVeu or LAVmal is cleaved with an appropriate restriction enzyme and that its portions are subsequently isolated, or • that the DNA from cells transformed with DNA for LAVEU or LAVmal according to European Patent Application No. 178 978 of September 17, 1985 , isolated. 35 DK 175819 B1 46 A method of producing the protein, glycoprotein or portion thereof according to any one of claims 9 to 11, which comprises the following steps: • that the DNA of claim 20 is expressed in a cell transformed therewith and that! The protein, glycoprotein or portion thereof is subsequently isolated, or • the protein, glycoprotein or portion thereof is synthesized in homogeneous solution by successively condensing successive amino acids or peptide fragments in an appropriate order. in